Delivery of various biomaterials including nucleic acids, proteins, cells, pharmaceutical agents and diagnostic agents has been a focus of intense research. Gene therapy is generally understood to refer to techniques designed to deliver nucleic acids, including antisense DNA and RNA, ribozymes, viral genome fragments and functionally active therapeutic genes into targeted cells (Culver, 1994, Gene Therapy: A Handbook for Physicians, Mary Ann Liebert, Inc., New York, N.Y.). Such nucleic acids can themselves be therapeutic, as for example antisense DNAs that inhibit mRNA translation, or they can encode, for example, therapeutic proteins that promote, inhibit, augment, or replace cellular functions. Success of gene therapy can be measured by ability to manipulate the rate and quality of gene delivery to an organism in need.
A serious shortcoming of current gene therapy strategies, including both ex vivo and in vivo gene therapy methods, is the inability of previously described vector and delivery system combinations to deliver nucleic acids efficiently into the interior of cells of a targeted population.
Virus vectors are generally regarded as the most efficient nucleic acid delivery vectors. Recombinant replication-defective virus vectors have been used to transduce (i.e., infect or transfect) animal cells both in vitro and in vivo. Such vectors include retrovirus, adenovirus, adeno-associated virus, and herpes virus vectors. Although they are highly efficient for gene transfer, one major disadvantage associated with the use of virus vectors is the inability of many virus vectors to infect non-dividing cells. Another serious problem associated with the use of virus gene vectors is the potential for such vectors to induce an immune response in a patient to whom they are administered. Such an immune response limits the effectiveness of the virus vector, since the patient's immune system rapidly clears the vector upon repeated or sustained administration of the vector. Furthermore, insertion of a gene into the genome of a cell by a virus vector can induce undesirable mutations in the cell. Other problems associated with virus gene vectors include inability to appropriately regulate gene expression over time in transfected cells, toxicity and other side effects caused by delivery of virus vectors to human tissues (e.g., liver damage and myocarditis), and potential production and transmission to other humans of harmful virus particles.
Furthermore, virus gene vectors, as used in prior art methods, frequently cannot be delivered to a selected tissue in a specific, localized manner. Instead, many prior art methods of administering virus vectors result in vector being dispersed systemically to tissues which adjoin, or are in fluid communication with, the desired target tissue. The inability of such methods to localize virus vectors reduces the utility of the methods, because a non-localized virus vector can transfect unintended tissues, elicit immune responses, be rapidly excreted from the body, or otherwise have a diminished transfection ability. A significant need exists for methods of delivering virus vectors in a localized manner.
Virus vectors can be used as vehicles to deliver proteins and other therapeutic molecules to the cells which the virus vectors transfect. Such proteins and other therapeutic molecules can be incorporated passively and non-specifically into virus vector particles. Alternatively, virus vectors specifically incorporate fusion proteins comprising a protein having a polypeptide viral packaging signal fused therewith.
Even though virus vectors have been widely used in experimental gene therapy protocols and human studies (Feldman et al., 1997, Cardiovasc. Res. 35:391-404; Roth et al., 1997, J. Natl. Cancer Inst. 89:21-39), none of these vectors has proven to be efficacious for a virus vector-mediated gene therapy. It has been hypothesized that the shortcomings of adenovirus vectors have been due, at least in part, to limited transgene expression resulting from the immune response of the host individual and cytotoxic effects toward organs of the host individual (Smith et al., 1996, Gene Ther. 3:190-200; Tripathy et al., 1996, Nat. Med. 2:545-549; Nabel et al., 1995, Gene Ther. Cardiovasc. Dis. 91:541-548). Other researchers have concentrated their efforts on mutating adenovirus vectors to render them relatively less immunogenic and toxic.
In addition to the low efficiency of a virus vector uptake exhibited by most cell types and low levels of expression of the gene constructs delivered by virus vectors, many targeted cell populations are found in such low numbers in the body that the efficiency of transfection of these specific cell types is even further diminished. Thus, there is a need for gene therapy methods which can be used to efficiently deliver virus vectors to targeted cell populations. Others working in the field have concentrated on attempting to specifically target adenovirus vectors to a particular cell type, for example, by attaching a specialized receptor ligand to the vectors (Tzimagiorgis et al., 1996, Nucl. Acids 24:3476-3477).
To be useful to gene delivery, a virus vector must be delivered to its target cells in a form in which the biochemical components of the virus vector retain their function. Specifically, the virus vector must retain the capacity to bind to target cells, to transfer a nucleic acid carried by the vector into the interior of the cell, and, in some circumstances, to catalyze chemical reactions involving that nucleic acid within the cell (e.g., reverse transcription, integration into the host cell genome, or promoting transcription of gene elements on the nucleic acid). Thus, it is important that the virus vector is administered to a patient without being exposed to chemically harsh or biochemically inactivating conditions. Further, many matrices are not compatible for contacting with virus vectors. Ideally, a matrix in or on which a virus vector is disposed should be biodegradable, and in a form suitable to use in surgical and therapeutic interventions.
Others have demonstrated enhancement of transfection effected by combining adenovirus vectors with polylysine or cationic lipids to form soluble virus vector complexes (Fasbender et al., 1997, J. Biol. Chem. 272:6479-6489). However, such virus complexes still exhibit many of the disadvantages described herein which are characteristic of virus vectors, including a short duration of the period during which the virus vector is available to contact with the desired tissue.
One approach to the biomaterial delivery is to coat a medical device with a composition comprising the biomaterial from which the biomaterial is released (e.g., U.S. Pat. No. 6,143,037 to Goldstein et al. and references therein). The problem with such coatings is that that they can invoke acute or chronic inflammatory responses due to the nature of coatings (see Lincoff et al., J. Am. Coll. Cardiol., 29, 808.16 (1997)). The nucleic acid delivery from coatings has also been problematic due to the limited ability to transfer a nucleic acid efficiently into a targeted cell population and achieve a high level of expression of the gene product in vivo. Further, current methods do not provide a sufficiently strong connection between the biomaterial and the delivery vehicle. For example, incorporating plasmid DNA into a collagen sponge and implanting it in bone can successfully deliver the nucleic acid but most of the DNA escapes in a very short time (e.g., less than one hour) (see Bonadio et al., Nat. Med. 1999, 5(7):753-9). Other known methods do not provide a sufficient release of biomaterial by ways other than biodegradation of the matrix, which may be too inefficient.
There have been attempts to solve these problems by incorporating biodegradable regions in a coating. See, for example, U.S. Pat. No. 6,639,014 to Pathak et al. disclosing a controlled release delivery of a biologically active material incorporated in biodegradable hydrogels. However, this approach does not solve the problem of insufficiently tight connection between the coating and the surface coated.
Inventors have demonstrated previously that gene therapy vectors can either be attached to surfaces or contained within other delivery systems using affinity adaptors (or connectors), such as specific antibodies or recombinant proteins (e.g., receptor fragments) (see U.S. patent application Ser. No. 09/487,949 by Levy et al., U.S. Patent Application Publication No. 2003/0044408A 1 by Levy et al., and U.S. Pat. No. 6,333,194 to Levy et al.). Others have attempted to deliver charged bioactive agents to biological systems by reversibly binding charged bioactive agents to oppositely charged electrode surfaces, contacting the electrodes with the biological system, and thereafter relieving the charge on the electrode surface (e.g. U.S. Pat. Nos. 4,585,652 and 5,208,154). Such methods are severely limited by the necessity to have electrical leads connecting the electrodes to a power source and by the difficulty of effectuating the sustained release of the bioactive agent from the electrode surface. Thus, the usefulness of such compositions for a delivery of virus vectors to specific tissues is limited.
A critical need remains for compositions suitable to deliver biomaterial to desired tissues in a manner in which the period during which the biomaterial is administered is prolonged and immunogenicity associated with such administration is minimized. At the same time, such compositions should not adversely affect the biological activity of the biomaterial to be delivered (e.g., the transfecting efficiency of the vector). The compositions and methods of the invention described herein satisfy this need.
All references cited herein are incorporated herein by reference.